The serine proteases are a class of enzymes which includes elastase, proteinase 3, chymotrypsin, cathepsin G, trypsin, thrombin, prolyl oligopeptidase and others. A breakdown in the balance of protease/antiprotease activity has been implicated in the pathogenesis of numerous disease states.
For example, it is known that many human malignancies are associated with enhanced expression of proteases (Opdenakker et al., Cytokine, 4:251-258 (1992)). These proteases may be involved in growth, chemotaxis, endocytosis, exocytosis, blood coagulation, fibrinolysis and tissue invasion during metastasis of malignant cells (Eisenbrand. Synthesis, 1246-1252 (1996); Kazama, J.B.C. 270:66-72 (1995)). Specifically, plasmin, urokinase-plasminogen activator (uPA) and tissue plasminogen activator (tPA) and a tumor-associated trypsin show a substantial increase in activity in many malignancies.
Tumor-associated trypsin has been shown to participate in cancer cell-mediated degradation of extracellular matrix (Koivunen et al., Cancer Research, 51:2107-2112 (1991)) and has been implicated in tumor invasion. Human urinary trypsin inhibitors have been shown to prevent both the intravasation and extravasation step of tumor metastasis (Int. J. Cancer, 63:455-462 (1995)). Urokinase-plasminogen activator and tPA have also been shown to be important in tumor metastasis (Dowell, et al., Cancer Treatment Reviews, 19:283-296 (1993)). A guanidinobenzoatase has been implicated in cancer metastasis, cell migration and tissue remodeling (Poustis-Delpont C. et al., J.B.C., 269:14666-71 (1994); Stevens et al., Br. J. Cancer, 46:934-939 (1982); Ohkoshi et al., J. Max. Fac. Surg., 12:148-152 (1984); Poustis-Delpont et al., Cancer Research, 52:3622-3628 (1992); Stevens, Biol. Chem., 69:137-143 (1988)).
Increased plasmin activity has been directly correlated to increased plasminogen activator activity (Eisenbrand, Synthesis, 1246-1252 (1996)). Compounds capable of inhibiting plasmin, tumor-associated trypsin, guanidinobenzoatase or plasminogen activator represent potential drug candidates for the treatment of the various human malignancies.
Hepsin, a membrane associated serine protease, has been shown to activate human factor VII and to initiate a pathway of blood coagulation on the cell surface leading to thrombin formation (Kazama. J.B.C., 270:66-72 (1995)). It is believed that a variety of neoplastic cells activate the blood coagulation system, causing hypercoagulability and intravascular thrombosis via this and other pathways and that hepsin plays a role in their cell growth, maintenance and morphology (Torres-Rosada et al., Proc. Natl. Acad. Sci. USA, 90:7181-7185 (1993)). Hepsin is present at elevated levels in regions of active cell proliferation in animal models and anti-hepsin antibody has been shown to suppress the growth of human hepatoma cells in culture. Hepsin is also suspected to be a physiological inactivator of the tumor suppressor protein maspin.
Chymase is believed to be responsible for angiotensin I converting enzyme (ACE) independent activation of angiotensin II in the heart (Urata, et al., J.B.C., 265:2963-2968 (1997)). This event appears to play a role in the hypoxic or ischemic heart (Urata, et al., Am. J. Hyprtens., 9:277-284 (1996)). In addition, in the failing or compromised heart, human chymase is believed to be responsible for an alternate production of angiotensin II, which has been shown to take place in the presence of angiotensin converting enzyme (ACE) inhibitor.
A cause and effect relationship has been shown between endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in experimental animal models (Zhu, et al., J. Clin. Invest., 94:1163-1171 (1994)). Pulmonary hypertension is commonly associated with congenital heart defects, pulmonary diseases associated with chronic hypoxia, hepatic disorders and connective tissue disease. Increased pulmonary artery elastolytic activity associated with the monocrotaline-induced pulmonary hypertension model has been shown to be moderated by treatment with an elastase inhibitor (Ye, et al., Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1255-H1267 (1991); Cowan et al., J. Clin Invest., 97:2452-2468 (1996)). In some models, early inhibition of EVE activity largely prevented pulmonary vascular damage. Although, EVE has been shown to be sensitive to leukocyte elastase (LE) inhibitors, it is believed that it is a novel enzyme distinct from LE. Inhibitors of EVE may be useful in treating pulmonary vascular disease in infants, restenosis secondary to angioplasty, pulmonary hypertension myocarditis, bronchopulmonary dysplasia, myocardial necrosis after cardiac transplant, post-cardiac transplant coronary arteriopathy, atherosclerosis and reperfusion injury following myocardial infarct.
Prolyl oligopeptidase degrades oxytocin, neurotensin, substance P, thyrotropin releasing hormone, bradykinin, angiotensin II and vasopressin (U.S. Pat. No. 5,506,256; Tsutsumi, et al., J. Med. Chem., 37:3492-3502 (1994)). It is also believed to degrade amyloid precursor protein, and therefore is suspected to play a role in Alzheimer's disease.
Aprotinin, a pancreatic basic trypsin inhibitor, was first described by H. Kraut et al. in 1930 (J. Physiol. Chem., 192:1 (1930)). Aprotinin has been studied and characterized extensively over the last few decades. It is the active ingredient of Trasylol.RTM. (Miles Laboratories), a drug which is marketed for the treatment of diseases such as hyperfibrinolytic hemorrhage and traumatic-hemorrhagic shock, or acute pancreatitis. Aprotinin has been shown to be an inhibitor of serine proteases (Fritz et al., Drug Res. 33 (1):479-494 (1983)), and is considered to be a broad-specificity inhibitor. In addition to trypsin and chymotrypsin, aprotinin inhibits plasmin and several kallikreins. It is also used prophylactically to reduce perioperative blood loss and to reduce the need for blood transfusions, mainly in patients undergoing coronary artery bypass graft surgery.
Although aprotinin is generally well tolerated, its main adverse side effects include increased incidence of postoperative renal dysfunction and allergic reactions, in some cases leading to anaphylaxis, which has in some cases been fatal. Patients who have had prior exposure to aprotinin are at higher risk for anaphylaxis. These two main adverse effects are both due to the properties of aprotinin as a protein, problems which may be overcome with a small, low molecular weight, synthetic drug with a similar inhibition profile as aprotinin. Additionally, new synthetic drugs would eliminate the need for extracting aprotinin from bovine lung or for its combinatorial expression.
It is clear that a need exists for methods of treating and/or preventing serine protease mediated pathologies.